(a) Field of the Invention
The present invention relates to a method for detecting positions of samples applied onto a carrier being shifted in a definite direction, and more specifically to a sample detection method used for quantitative analyses of fractionated patterns of sera in the electrophoresis.
(b) Description of the Prior Art
In the electrophoresis, samples (sera) are applied onto a carrier made of cellurose acetate or the similar material and fractionated patterns of the samples are formed by electrically energizing the carrier. Then, the carrier is colored, discolored and made transparent, whereafter the fractionated patterns are subjected to quantitative analyses with a colorimeter. For automatic analyses of the samples with a colorimeter, the carrier is shifted between a light source and a photo detector, the carrier is stopped each time a sample applied onto the carrier is located right between the light source and photo detector, and the light source and photo detector are moved together in the direction perpendicular to the shifting direction of the carrier to scan the sample for carrying out photometry. Such a photometric apparatus requires a sample detector which precisely detects position of a sample on the carrier when it is located right between the light source and photo detector.
Recently, there has been developed an automatic electrophoretic apparatus so adapted as to carry out all the steps from application of samples onto a carrier to densitometry in electrophoresis. In this electrophoretic apparatus, samples are applied onto a carrier at constant intervals in the step of sample application. However, intervals between the samples are made different at the step of densitometry due to conditions at the intermediate steps, for example, difference in electrophoretic conditions and contraction of the carrier at the drying step. Though such variations in sample intervals are on the order of 2 mm, it is necessary to compensate the variations for carrying out densitometry correctly at the center of the fractionated patterns. Influence due to positional deviations will be more remarkable especially for samples applied onto later portions of the carrier.
In order to meet the requirements described above, there has recently been developed a sample detector having the construction illustrated in FIG. 1 wherein the reference numeral 1 represents a carrier on which electrophoretic patterns of samples are formed and which is shifted in the direction indicated by the arrow. The reference numeral 10 designates a first optical fiber group consisting of optical fibers 11 through 18 (eight optical fibers are shown as a non-limitative example) which have ends 11a through 18a so arranged as to make the lights emerging therefrom incident perpendicularly on the surface of the carrier and arranged in a row in the direction (x direction) perpendicular to the shifting direction of the carrier. At the other ends 11b through 18b of the optical fibers, there are arranged light emitting diodes 31 through 38 so as to correspond to the individual optical fibers. The reference numeral 20 denotes a second optical fiber group consisting of optical fibers 21 through 28 in the same number as those of the first optical fiber group. The individual optical fibers of the second optical fiber group have ends 21a through 28a which are arranged in a row opposite respectively to the ends 11a through 18a of the individual optical fibers of the first optical fiber group so as to receive the lights emerging therefrom. The other ends 21b through 28b of the individual optical fibers of the second optical fiber group are fastened in the form of a bundle. The reference numeral 40 represents a photo detector element arranged in the vicinity of the ends 21b through 28b of the bundled optical fibers, and the reference numeral 41 designates a preamplifier. The reference numeral 3 denotes a light source assembly consisting of a light source lamp 4, a lens system 5, a filter 6, a slit plate 7 and so on, and the reference numeral 8 represents a photo detector which composes a photometric system for carrying out photometry while scanning samples applied onto the carrier in the direction perpendicular to the shifting direction of the carrier. The ends 11a through 18a and 21a through 28a arranged in rows opposite to each other of both the above-mentioned optical fiber groups are positioned at a distance equal to a single pitch 1 of sample intervals as measured from the position of the photometric system. When a sample 2 on the carrier is placed at the position of the photometric system, the next sample 2' is therefore placed between the ends 11a through 18a and 21a through 28a of the optical fibers.
In the sample detector having the above-described construction, the carrier is shifted in the direction indicated by the arrow and at the same time the individual light emitting diodes 31 through 38 are glowed consecutively at a high speed at the timing illustrated in FIG. 2. After all the light emitting diodes have been glowed, they are glowed repeatedly in the sequence of 31, 32 through 38. The lights emitted from the light emitting diodes pass through the optical fibers 11 through 18, emerge from the ends 11a through 18a thereof, pass through the carrier, enters the corresponding optical fibers 21 through 28 of the second optical fiber group 20, transmit therethrough, emerge from the exit ends 21b through 28b thereof and are received by the photo detector element 40. Intensities of the received lights correspond to concentration of the sample applied onto the carrier, and the photo detector element provides, upon receiving the lights, outputs as exemplified in FIG. 3. These outputs are totalized by the preamplifier 41 which provides output as shown in FIG. 4. The preamplifier provides a high output when the light passes through a transparent portion of the carrier free from sample and has high intensity, whereas the output is lowered corresponding to concentration of sample when the light passes through a sample applied onto the carrier. Output corresponding to transparent portion free from sample is exemplified in FIG. 5A, whereas one corresponding to a sample is exemplified in FIG. 5B. Now, it is possible to preset a sample detection level as shown in the dashed lines in FIG. 5A and FIG. 5B respectively. Speaking more concretely, it is possible to preset a level a which is lower than all outputs when a sample is not detected and higher than at least one of outputs when a sample is detected. This level a is usable as a criterion for judging presence of a sample. A turning point from a time at which the outputs produced from the lights having passed through all the optical fibers are higher than the level a to another time at which an output produced from a light having passed through any one of the optical fibers is lower than the level a, just corresponds to the moment that a sample is just located between the ends of the optical fibers arranged in rows. Then, the time at which the outputs produced from the lights having passed through all the optical fibers become higher than the level a corresponds to the moment when the sample deviates from between the ends of the optical fibers. It is therefore possible to detect both the front and rear edges of a sample in this way. When presence of a sample is detected as described above, it is possible to stop the carrier to locate the sample correctly at the position of the photometric system in a definite time determined depending on the gaps as measured from the photometric system to the ends of the optical fibers.
In FIGS. 5 and 5B, the outputs are illustrated on an assumption that they are kept constant when a transparent portion free from sample of the carrier is located between the ends of both the optical fiber groups. In actuality, however, the outputs more or less vary due to the facts that intensities of the lights emitted from the individual light emitting diodes are different and that transmittance of the individual optical fibers is more or less different. Therefore, more adequate judgement can be done when outputs corresponding to the individual light emitting diodes are measured by glowing them before detecting samples, correct output levels for the individual optical fibers are stored on the basis of the measured values, and these levels are used as standard levels (corresponding to the level a shown in FIG. 5A and FIG. 5B) for judgement. Such measurements of output levels for the individual optical fibers permit sample detection with higher accuracy since they correct not only variations in intensities of the lights emitted from the individual light emitting diodes and transmittance of the individual optical fibers but to compensate variations of the entire detector system including drift of the electric circuit. Processing of the data obtained with the above-described sample detector is performed according to a computer program. FIG. 6 shows a block diagram of a data processing system for such a purpose, in which image signals subjected to photoelectric conversion by the photo detector element 40 are amplified by a preamplifier 41, whose outputs are converted by an A/D converter unit 42 into digital signals to be fed and stored into a computer 43. The reference numeral 44 represents an oscillator which is operated with an image detection command from the computer 43 and whose outputs are converted by a converter 45 into signals for glowing the individual light emitting diodes consecutively and fed into a driver circuit 46 which functions to glow the individual light emitting diodes 31, 32, . . . consecutively at definite time intervals. On the other hand, the outputs from the oscillator are fed also into a one-shot multivibrator, which in turn provides an output as A/D conversion command to the A/D converter unit 42. The A/D converter unit 42 creates an A/D conversion end signal which is fed into the computer 43 for taking the data thereinto at that time. Since the light emitting diodes are glowed consecutively, it is possible to make the individual light emitting diodes correspond to the data in a relationship of 1:1 so as to identify each item of the data corresponding to each of the light emitting diodes.
FIG. 7 shows a time chart clarifying the signals at the respective stages of the block diagram illustrated in FIG. 6. In FIG. 7, the reference symbol (A) represents signals transmitted from the oscillator and the reference symbol (B) designates outputs from the one-shot multivibrator which is triggered at the falling ends of the transmission signals A. The reference symbols (C.sub.1), (C.sub.2) through (C.sub.8) denote driving signals for the light emitting diodes created by the converter using the transmission signals A and to be used for glowing the light emitting diodes. Upon glowing of the light emitting diodes, the lights are received by the photo detector element to produce image signals, which are amplified by the preamplifier to prepare outputs D. It is possible to feed output signals at the glowing timing of the light emitting diodes into the computer by feeding these output data into the computer at the timing determined on the basis of the conversion end signal E created by the A/D converter unit.
When positions of samples are detected with a sample detector having the above-described construction, a sample at a high concentration provides a broad fractionated patterns, i.e., so widened in the shifting direction of the carrier as to be overlapped with these of the adjacent sample applied onto the carrier as illustrated in FIG. 8 and FIG. 9. In FIG. 9, samples 2'a, 2"a and 2'''a at concentrations higher than that of sample 2a have sample widths gradually broadened. Therefore, sample positions 2b, 2'b, 2"b and 2'''b corresponding to the standard level a for detecting sample position are different from one another. In other words, positions of the samples to be detected are different depending on concentrations of samples, resulting in an undesirable effect. In case of a sample at a very high concentration such as the sample 2'''a, sample width becomes broad enough to overlap with the neighboring sample 2""a as shown in FIG. 8, thereby making the overlapped area 2c at a concentration higher than the standard level (on the side of the dark level) and making it impossible to detect position of the sample. In case of a sample at a low concentration as shown in FIG. 9, in contrast, the area even at the highest concentration in close to the standard level, thereby making sample detection unstable.